Douglas E. Williams

Identification of a ligand for the c-kit proto-oncogene

We report the purification and N-terminal amino acid sequence of a novel mast cell growth factor, termed MGF, from the supernatants of a murine stromal cell line. A panel of interleukin 3-dependent cell lines were screened for responsiveness to partially purified MGF in [3H]thymidine incorporation assays; proliferative stimulation of these cells in response to MGF correlated with expression of mRNA for the c-kit protooncogene. MGF was shown to be a ligand for c-kit by cross-linking 125I-labeled MGF to c-kit-expressing cells with subsequent immunoprecipitation of the complex with antiserum specific for the C-terminus of c-kit. This establishes MGF as a ligand for the c-kit protein.

Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms

We have previously reported the identification of a novel mast cell growth factor (MGF) that was shown to be a ligand for c-kit and is encoded by a gene that maps near the steel locus on mouse chromosome 10. We now report the cloning of cDNAs encoding the MGF protein. The MGF protein encoded by this cDNA can be expressed in a biologically active form as either a membrane bound protein or as a soluble factor. The soluble protein promotes the proliferation of MGF-responsive cell lines and, in the presence of erythropoietin, stimulates the formation of macroscopic [corrected] erythroid and multilineage hematopoietic colonies.

Molecular cloning of a ligand for the flt3 flk-2 tyrosine kinase receptor: A proliferative factor for primitive hematopoietic cells

Cloning of a ligand for the murine flt3/flk-2 tyrosine kinase receptor was undertaken using a soluble form of the receptor to identify a source of ligand. A murine T cell line, P7B-0.3A4, was identified that appeared to express a cell surface ligand for this receptor. A cDNA clone was isolated from an expression library prepared from these cells that was capable, when transfected into cells, of conferring binding to a soluble form of the flt3/flk-2 receptor. The cDNA for this ligand encodes a type I transmembrane protein that stimulates the proliferation of cells transfected with the flt3/flk-2 receptor. A soluble form of the ligand stimulates the proliferation of defined subpopulations of murine bone marrow and fetal liver cells as well as human bone marrow cells that are highly enriched for hematopoietic stem cells and primitive uncommitted progenitor cells.

Mast cell growth factor maps near the steel locus on mouse chromosome 10 and is deleted in a number of steel alleles

Many spontaneous, chemical-induced, and radiation-induced dominant white spotting (W) and steel (Sl) mutations have been identified in the mouse. W and Sl mutations have similar phenotypic effects including deficiencies in pigment cells, germ cells, and blood cells, Numerous studies have suggested that W acts within the affected cell while Sl instead exerts its effects in the extracellular environment. Recent findings demonstrating that W encodes the c-kit proto-oncogene, a tyrosine kinase membrane receptor, have suggested that Sl encodes a ligand for c-kit. In the accompanying article we report the identification and purification of mast cell growth factor (MGF), a c-kit ligand. Here we describe the cloning of sequences encoding MGF. Furthermore, we show that Mgf maps near Sl in the distal region of mouse chromosome 10 and is deleted in a number of Sl alleles. These findings strongly support the notion that Sl encodes the mast cell growth factor.

The steel factor

Steel factor (SLF) is a recently identified growth factor which is the gene product of the murine Steel locus and a ligand for the c-kit tyrosine kinase receptor, the product of the dominant white spotting locus (W). Defects at these genetic loci result in aberrant melanocyte, germ cell, and hematopoietic development. Both the receptor (c-kit) and the ligand (SLF) have been shown to undergo tissue-specific mRNA splicing to produce distinct isoforms which have unique biological functions. As predicted by the phenotype of these mutations, SLF influences the growth and differentiation of melanocytes, primordial germ cells, and a broad spectrum of cell types in the hematopoietic progenitor and stem cell hierarchy. SLF has also been shown to have effects on hematopoietic lineages not predicted by defects seen in the Steel mouse.

Steel Factor Is Required for Maintenance, But Not Differentiation, of Melanocyte Precursors in the Neural Crest

Skin melanocytes are derived from neural crest cells that migrate into the dermis during embryogenesis. Two mouse mutants, Steel and White dominant-spotting, which have defects in melanocyte production, have recently been shown to have deletions in the genes that code for a new growth factor, steel factor (SLF), and its receptor, respectively. Here, we have investigated the role that SLF plays in melanogenesis using cultures of mouse neural crest and found that its primary action is the maintenance of melanocyte precursors. It has no effect on the final stage of melanocyte differentiation, the production of melanin, which appears to require an additional factor whose action is mimicked by the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate).

Comparative effects in vivo of recombinant murine interleukin 3, natural murine colony-stimulating factor-1, and recombinant murine granulocyte-macrophage colony-stimulating factor on myelopoiesis in mice.

Purified murine colony-stimulating factors (CSF) recombinant interleukin 3 (IL-3), natural CSF-1, and recombinant granulocyte-macrophage (GM) CSF were assessed in vivo for their effects on BDF1 mouse bone marrow and spleen granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells in untreated mice and in mice pretreated with purified iron-saturated human lactoferrin (LF). The CSF and LF preparations did not contain detectable endotoxin (less than 0.1 ng). Mice pretreated with LF were more sensitive to the effects of CSF. In mice pretreated with LF, 2,000 U IL-3 or 20,000 U CSF-1 significantly enhanced the cycling status and absolute numbers of all progenitors, whereas 20,000 U GM-CSF significantly increased the cycling status of CFU-GM and CFU-GEMM, but had no effect on cycling of BFU-E or on numbers of any of the progenitors. The effects of CSF in mice pretreated with LF were not mimicked by 0.1-100 ng E. coli lipopolysaccharide.

The production of myeloid blood cells and their regulation during health and disease

The regulation of myelopoiesis in vivo most likely entails a complex set of interactions between cell-derived biomolecules and their target cells: hematopoietic stem and progenitor cells and accessory cells. Stimulating and suppressing factors have been characterized through in vitro studies, and their mechanisms of action in vitro and in vivo have begun to be elucidated. Among those factors being studied are the hematopoietic colony-stimulating factors (CSF): interleukin-3 (multi-CSF), granulocyte-macrophage-CSF, granulocyte-CSF, and macrophage-CSF; other molecules include erythropoietin, B-cell-stimulating factor-1, interleukin-1, interleukin-2, prostaglandin E, leukotrienes, acidic ferritins, lactoferrin, transferrin, the interferons-gamma, -alpha, and -beta, and the tumor necrosis factors-alpha and -beta (lymphotoxin). These factors interact to modulate blood cell production in vitro and in vivo. The proposed review characterizes these biomolecules biochemically and functionally, including receptor-ligand interactions and the secondary messengers within the cell which mediate their functional activity. The production and action of the molecules are described under conditions of hematopoietic disorders, as well as under normal conditions. Studies in vitro are correlated with studies in vivo using animal models to give an overall view of what is known about these molecules and their relevance physiologically and pathologically.

Effects of PIXY321, a granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein, on chemotherapy-induced multilineage myelosuppression in patients with sarcoma.

PURPOSE To evaluate the clinical safety and ability of PIXY321, a novel fusion protein of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), to ameliorate chemotherapy-induced multilineage myelosuppression. PATIENTS AND METHODS PIXY321 was administered by subcutaneous injection twice daily (25 to 1,000 micrograms/m2/d) over 14 days to 24 chemotherapy-naive patients with sarcoma in a phase I/II study. Three weeks from the initiation of PIXY321, the first cycle of chemotherapy with cyclophosphamide, doxorubicin, and dacarbazine (DTIC) (CyADIC) was administered over 3 days. Four weeks later, a second cycle of CyADIC was administered, followed by 14 days of PIXY321. RESULTS Treatment with PIXY321 was well tolerated. Local skin reactions and constitutional symptoms were the main side effects. The dose-limiting toxicity was not encountered; however, headache and fatigue were more frequent at the highest dose (1,000 micrograms/m2). PIXY321 before chemotherapy elicited a modest increase in the WBC count (consisting mainly of mature neutrophils), platelets, and corrected reticulocyte counts (all P < .001). Following chemotherapy, PIXY321 at effective doses (500 to 1,000 micrograms/m2/d), significantly reduced both the degree (mean nadir, 70 v 310/microL; P = .016) and duration (mean days < 500/microL, 6.6 v 3.9 days; P = .002) of neutropenia. Cumulative thrombocytopenia was not observed during the first two cycles of CyADIC (mean nadir platelet count, 103 v 95 x 10(3)/microL, in cycles no. 1 and 2, respectively; P = NS). Compared with our historic control data, the mean nadir platelet count in cycle no. 2 was significantly higher after PIXY321 (1.7-fold, P < .05) than with CyADIC alone or with GM-CSF support. There was a suggestion for a dose response, since the mean percentage change in nadir platelet values from cycle no. 1 to cycle no. 2 increased with the PIXY321 dose (P < .02), with the peak effect observed at 750 micrograms/m2/d. CONCLUSION These results suggest a potential clinical role for PIXY321 in attenuating the cumulative multilineage hematopoietic toxicity of chemotherapy.

biology and potential clinical applications of flt3 ligand

&NA; The flt3 ligand is a member of a small family of growth factors that stimulate the proliferation of hematopoietic cells. Other members of this family include Steel factor (also known as mast cell growth factor, stem cell factor, and kit ligand) and colony-stimulating factor 1. These proteins function by binding to and activating unique tyrosine kinase receptors. Both flt3 ligand and Steel factor stimulate the proliferation of early progenitor or stem cells. Neither of these factors exhibits much biologic activity by itself, but each factor can synergize with a wide range of other colony-stimulating factors and interleukins. One major difference between the two factors appears to be their effect on mast cells, which Steel factor stimulates, but flt3 ligand does not. Although flt3 ligand and Steel factor each act on early hematopoietic cells, differences in their activities suggest that they are not redundant and are both required for normal hematopoiesis. There are a number of clinical settings in which the flt3 ligand may prove quite useful.